development of biosensors -...

Development of biosensors

Prof. dr. habil. Arūnas Ramanavičius (1)Assoc. prof. dr. Almira Ramanavičienė (2)

1. Department of Physical Chemistry Faculty of Chemistry, Vilnius University,

2. Center of NanoTechnology and Materials science, Faculty of Chemistry, Vilnius University, Naugarduko 22, 2006 Vilnius, Lithuania;

Lithuania (Lietuva)

NanoTechnas

Capital: Vilnius

3.5 Mln. People.

Vilnius University

Was founded in 1579

NanoTechnas

[taisyti ] Strukt ūra

Center of NanoTechnology and Materials science - NanoTechnas

Faculty of Chemistry, Vilnius University

Center of NanoTechnology and Material science -

NanoTechnas

Center of NanoTechnology and Material science –NanoTechnas established within 2005-2008

NanoTechnasestablished within 2005-2008

Some Equipment in NanoTechnas,

•Bio – AFM (Veeco)• SPR device (ECO-Chemie);• QCM+EQCM (MacTex); Potentiostat/Galvanostat (ECO-Chemie);

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Outline

• Introduction – Center of Nanotechnology and materials science –NanoTechnas; Our major research directions; • Clasification of biosensors:

• Catalytic biosensors,• Immunosensors,• DNA sensors, sensors,• Molecularly imprinted polymer based sensors,• Biofuel cells;

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• Biofuel cells;•Some historical aspects, interconnections: From catalytic biosensors towards biofuel cells;• Redox enzymes suitable for construction of biosensors and biofuel cells; some limitations and advantages;• Immobilization methods suitable for the immobilization of redox enzymes;• Application of biosensors and biofuel cells, major types and major directions; • Implantable biosensors and biofuel cells, some suitable materials;• Concluding remarks.

Some our research directions

Sensors and Biosensors

Fuel cells and Biofuel cells

Catalytic DNA Immuno- Moleculary Catalytic

Enzymatic

Sensors

DNA

Sensors

Immuno-

Sensors

Moleculary imprinted polymer based

Sensors

Biomedicine: Implants; Drug delivery systems

Introduction // Sensors // Lab on the chip

Do we need sensors?

Why do we need sensors?

Sensors // What they are??

Introduction // Some Challenges in Sensorics and

Biosensorics

Sensors Today and Tomorrow

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Classification of Biosensors

Sensors and Biosensors

Catalytic DNA Immuno- Moleculary

Fuel cells and Biofuel cells

Catalytic

Sensors

Enzymatic biosensors

DNA

Sensors

Immuno-

Sensors

Moleculary imprinted polymer based

Sensors

Some analytical signal detection methods in biosensors

Optical Surface Plasmon Resonance // Elipsommetry

Photoluminescence

Microgravimetric

ElectrochemicalElectrochemical

Potentiometric

Amperometric

Constant potential based methods

Potentiodynamic methods

Cyclic voltammetry

Pulsed potential based amperommetry

Electrochemical impedance spectroscopy

Part of biological recognition (catalyst)

Signal transducer

AnalyteProducts

Biosensors

Registration

device

Fundamental task –stabile and effective attachment of biological objects on the surface of signal transducer.

In electrochemical biosensors electron transfer is requested

Glc

Gluconolactone 2H O 2 2

GODM

M

ox.

red.

2e-2e

-2e

-

Ele

ctr

ode

O 2

2H O2

+

M =PM S; K3[Fe(CN)6]; ferocene derivates …

Glc

Gluconolactone

GDHM

M

ox.

red.

2e-2e

-2e

-

Ele

ctr

ode

PQQ-

M =PM S; DCPIP; BQ

The most promising Enzymes, since has “well established intrinsic electrical circuit” and are able to transfer electrons

Enzymes in design of catalytic biosensors

ferocene derivates …

Glc

Gluconolactone

GDHM

M

ox.

red.

2e-2e

-2e

-

Ele

ctr

ode

2NADH

2NAD+

NAD-

Alcohol

Aldehyde

HQ-ADH

e-

2e-

Ele

ctr

ode

PQQ

e-

e-

e-

e-

hem

e chem

e c

hem

e c

hem

e c

directly to conducting surfaces.

Substrate Product

Substrate Product

Substrate Product Substrate Product Substrate Product

Oxidases – mostly used enzymes in catalytic biosensor design

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O2 H2O2 Medox Medred O2 H2O2 Medox Medred

Ramanavicius A. (2007) Amperometric Biosensor for the Determination of Cre atine , Analytical and Bionalytical Chemistry 387:1899–1906.

Substrate Product Substrate Product

Substrate Product

Substrate Product

Substrate Product

NAD-dependent dehydrogenasesin catalytic biosensor design

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O2 H2O2 Medox Medred


Ramanavicius A. Ramanaviciene A, Malinauskas A. (2006) Electrochemical Sensors Based on Conducting polymer – Polypyrrole (R eview)”Electrochimica Acta 51, 6025-6037.

Some Red-Ox Mediators

N

N

CH3

+

2 e-+

- 2 e- N

N

CH3

HDerivatives of Phenazine

N

R

N

Derivatives of Os-bipyridine complex

Fluorenone

NO2

NO2

O2N

O

N

HCH3

Derivatives of benzodiazepine

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Some RedOx MediatorsFe+++

-R

e-+

- e--

Derivatives of Ferrocene

Fe++

-R

-

N

NOs+3

N

N

R

N

N

CH3

CH3

N

O

OH

HO

Derivatives of quinones

Prussian Blue

Fe+34[Fe+2(CN)6]3

� First generation � Second generation � Third generation

Substrate Product Substrate Product

Electron transfer in biosensors

and Biofuel cells

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Substrate Product


Potential range

E / mV vs. NHE-100 0 +100+200 +300+400 +500 +600 +700 +800+900-200-300

NAD+ /NADH

EЎ'pH7 = -320 mV vs. NHE

O2/H2 O

EЎ'pH7 = +815 mV vs. NHE, [O2]=0.26 mM

-400

H+/H2 EЎ'pH7 = -420 mV vs. NHE

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cytochromeshydrogenases complex I laccasebilirubin oxidase

peroxidases

anode reactions cathode reactions

cytochrome c oxidase

Polypyrrole

Hemas-c Hemas-c

Hemas-cI IIeeee

e

eHeme-c Heme-c

Heme-c

Substrate Product

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PQQ Hemas-c

I II

IIIActo r. Aldehidas

eee

e

Ethanol

Heme-c

Ramanavi čius A., Habermüller K. Csöregi, E., Laurinavi čius V., Schuhmann. W. (1999) Polypyrrole entrapped quinohemoproteinalcohol dehydrogenase. evidence for direct electron transfer via conducting polymer chains, Analytical Chemistry, 71, 3581-3586.

Razumien ė J., Niculescu M., Ramanavi čius A., Laurinavi čius V., Csöregi E. (2002) Direct Bioelectrocatalysis at CarbonElectrodes Modified with Quinohemoprotein Alcohol Dehydrogenase from Gluconobacter sp. 33, Electroanalysis, 14, 43-49.

Aldehyde of Acetic Acid

Major immobilization methodsused for design of biosensors

�Adsorption;

� Covalent attachment;

� Cross-linking with chemical agents;

�Application of SAM’s followed by covalent attachment;

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�Application of SAM’s followed by covalent attachment;

� Entrapment within polymers.

Conducting polymers might be very useful: (i) for modification, (ii) and for protection of biosensor surfaces. (2nd lecture)Ramanaviciene A., Ramanavicius A. (2004) Affinity sensors based on nano-structured p-p conjugated polymer polypyrrole. In: D.W. Thomas (ed.) Advanced Biomaterials for Medical Applications. Kluwer Academic Publishers, Netherlands, pp. 111-125.

Some problems with direct ET due to

random orientation (e.g. on carbon)

Adsorption

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O OH OO

OHOOH O OH O

OOHO

OH O OH O

OOHO

OH O OH O

OOHO

OH

NHBio H2N Bio

O

HC-(CH2)3-C

O

H

=CH-(CH ) -CH= N NBio Bio

Immobilization

methods cross-

linking

+O

C-(CH ) -CO

+

Pentandiol (Glutaraldehyde)

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NH2Bio H2N Bio =CH-(CH2)3-CH= N NBio Bio+ HC-(CH2)3-C

H +

Bio

BioBio

Bio

Functionalised thiol modified gold

offers ordered orientation of enzymes

Almost 100% of enzyme molecules in

Self assembled monolayers (SAM’s)

Additional Resistance

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Almost 100% of enzyme molecules in

well oriented and may directly

electronically contact with the electrode

Resistance

Electrochemically polymerisableCompounds with Red-Ox Mediators

Fc

OH

ElectropolymerisationFc

OH

Fc

OH

Fc

OHn

Ferrocenephenol (FP)

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Electrochemical polymerisation

(FP1)

FcOH

N-(4-Hydroxybenzylidene)-4-ferrocenylaniline

N

FcOH

2-Ferrocenyl-4-nitrophenol

NO 2

(FcNO2)

Part of biological recognition

Signal transducer

AnalyteProducts

Biosensors

Registration

device

Ramanavicius A. Malinauskas A. Ramanaviciene A. (2004) Catalytic biosensors based on conducting polymers. In: D.W. Thomas (ed.) Advanced Biomaterials for Medical Applications. Kluwer Academic Publishers, Netherlands, pp. 93-109.

GDH

GDH

Glucose

Glucono-lactone

Glucose

PQQ

PQQ

PMSoks.

PMSred.

COO -ICOO -I

COO -I

COO -I

COO -I

COO -I COO -

ICOO -I

COO -I COO -

ICOO -I

COO -ICOO -

ICOO -ICOO -

ICOO -ICOO -

ICOO -I

COO -I

COO -I COO -

ICOO -I

COO -ICOO -

ICOO -ICOO -

ICOO -ICOO -

ICOO -I

COO -I

Glucose

Ascorbic a. Urate- -PMS mediated biosensor.

Based on PQQ-GDH entrapped within

conducting polymerProtection from interfering chemicals, by over-oxidized Ppy layer Catalytic conversion of analyte GDH

e-Glucono-lactone

PMSred.PMSoks.

Pt

analyte

Transfer of electrons from enzyme to conducting surface by diffusing “electron shuttles”

Ramanavicius A. (2000) Electrochemical study of permeability and charge-transfer in polypyrrole

films, Biologija, 2, 64-66.

Laurinavicius V., Razumiene J., Ramanavicius A., Ryabov A.D., (2004) Wiring of PQQ–dehydrogenases,

Biosensors & Bioelectronics 20 (6), 1217-1222.

Enzymatic Biosensors and Biofuel cells

Ramanavicius A. Kausaite A., Ramanaviciene A, (2005) Biofuel cell based on direct bioelectrocatalysis, Biosensors and Bioelectronics,. 20: 1962-1967.

Ramanavicius A., Kausaite A., Ramanaviciene A., (2006) Potentiometric Study of Quinohemoprotein Alcohol Dehydrogenase Immobilized on the Carbon Rod Electrode, Sensors and Actuators B: Chemical, 113, 435-444.

Potentials of (1) MP-8 functionalized cathode as

function of hydrogen peroxide concentration; (2) MP-

8/GOx functionalized carbon rod electrode as a function

of hydrogen peroxide concentration; (3) MP-8/GOx

functionalized carbon rod electrode as a function of

glucose concentration; (4) QH-ADH functionalized

anode as a function of ethanol concentration.

Investigations performed in 50 mM Na acetate solution,

pH 6,

Schematic representation of glucose oxidase coating bypolypyrrole initiated by catalytic action of this enzyme.

Polypyrrole Coated Glucose Oxidase Nanoparticles

Schematic representation of application of polypyrrole coated glucose oxidase nanoparticles in PMS mediated biosensor design.Ramanavicius A., Kausaite A., Ramanaviciene A. (2005) Polypyrrole Coated Glucose Oxidase Nanoparticles for Biosensor Design, Sensors and Actuators B-Chemical 111-112, 532-539. /// Ramanavicius A., Kausaite A., Ramanaviciene A., Acaite J., Malinauskas A., (2006) Redox enzyme – glucose oxidase –

initiated synthesis of polypyrrole Synthetic Metals, 156, 409-413

Activity of entrapped enzymes

O2 H2O2

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Ramanavicius A., Kausaite A., Ramanaviciene A. (2005) Polypyrrole Coated Glucose Oxidase Nanoparticles for Biosensor Design, Sensors and Actuators B-Chemical 111-112, 532-539.

Signal transducer

Part of biological recognition

AnalitėAnalitėAnalyteAnalyte Analyte

Registration

deviceFundamental task – stabile and effective attachment of biological objects on the surface of signal transducer and efficient signal transduction

Ramanaviciene A., Ramanavicius A. (2004) Affinity sensors based

on nano-structured p-p conjugated polymer polypyrrole. In: D.W.

Thomas (ed.) Advanced Biomaterials for Medical Applications. Kluwer Academic Publishers, Netherlands, pp. 111-125.

Generation of immune response

Blood

Antibodies against infection

Blood

Antibody

TIME: 2:00

Viewing Window

YYY

YYYY

YYY

YYYY

YYYY

Y

Y

Positive signal

Control

V

i

r

u

s

a

s

Integrase

Lipid layer

Reverse transcriptase

p24

Structure of Retroviruses

Human immuno-deficit virus

gp51/gp30RNR

p15

Proteins applied in construction of immunosensors

BLV

gp51gp30

Lipid bilayer of the envelope

Adsorbtionand invasion

Back transcriptase

Mature virus

Budding

Integrase

Genomic RNR

Replication of retroviruses

RNA

Back transkription

Nukleproteinin complex

Intranuklear transportand integration

Integrated DNAof provirus

Cellular DNA

Expresion of viral genome

mRNA

RNR

Protein syntezis, procesing and

assembly of virion

R

Signal transducer

Target DNA

DNA-sensor

Registering device

Structure of DNA

Detection of interaction between ssDNA and target DNA

Ramanavičienė A. Ramanavičius A. (2004) Pulsed amperometric detection of DNA with an ssDNA/ polypyrrole modified

electrode Journal of Analytical and Bioanalytical Chemistry, 2004; 379 (2): 287-293.

DNA Determination

•

• Multiplication

200 mV/s

-100

0

100

200

300

0 100 200 300 400 500 600 700 800

Detection by cyclic voltammetry

Before

-400

-300

-200

-100

p rie š in k u b a v imą p o in k u b a v im o

After incubationBefore incubation

Before incubation

10

15

20

25

Detection by potential-pulse amperometry

-25

-20

-15

-10

-5

0

5

0 20 40 60 80 100 120

Before incubation

After incubation

Before incubation

Imag

inar

y Im

peda

nce

(Z")

, KO

hm

80

100

120

Electrochemical impedance spectroscopy

Real Impedance (Z'), KOhm

0 20 40 60 80 100 120

Imag

inar

y Im

peda

nce

(Z")

, KO

hm

0

20

40

60 i ii

Before incubation

Ramanavicius A., Kurilcik

N., Jursenas S.,

Finkelsteinas A.,

RamanavicieneA. (2007)

Conducting Polymer

Based Fluorescence

Quenching as a new

Approach to Increase the

Selectivity of Selectivity of

Immunosensors

Biosensors and

Bioelectronics, 23, 499-

505.

Ramanaviciene A.,

Ramanavicius A. (2004)

Towards the hybrid

biosensors based on

biocompatible conducting

polymers. In: Shur M.S.,

Zukauskas A. (eds.) UV

Solid-State Light Emitters

and Detectors. Kluwer

Academic Publishers,

Netherlands, pp. 287-296.

Quantum Dots

• • 1 – 10 nm size, 100 – 1000 atoms• • Materials: conductors/semi-conductors• (CdS, CdTe, CdSe, ZnSe, etc. Other materials,

Au, Si, Ge, etc.)• • Electro-positive holes // electron capture• – Color shift• – Color shift• – Conductivity change

Surface Plasmon Resonance

Based Biosensors

Hardware setupHardware setup

Example

ANTIBODY – ANTIGEN

INTERACTION

+Kass

+kdiss

Antibody Antigen complex Ab:Ag

Theory of SPRTheory of SPR

Practical set-up of the hemi-

cylinder


Illustration of an interaction

Gold

α α

β β

Sensorgram: Sensorgram: angle (mangle (m°°) vs time (s)) vs time (s)

β β

SPR plot:SPR plot:Reflectivity (%) vs angleReflectivity (%) vs angle

The Kretschmann configuration

Lightreflection Intensity

SPR angleSPR angle

Intensityin %

Angle of incident light

within the range of total internal reflection, TIF

Light Intensityin %

Angle of incident lightAngle of incident light

62 ° 78 °

Dynamic range of 4000 m°

Light Intensityin %

Use of the spindle


Typical SPR measurement

• Baseline

• Association• Association

• Equilibriu

m

• Dissociatio

n

• Regenerati

on

Theory of SPRTheory of SPR

Surface Plasmon Resonance

• Multiplication

Prof. habil. dr. Arūnas Ramanavičius

Center of NanoTechnology and Materials science -NanoTechnas,

Dėkoju už dėmesį!

Thank you for attention!

Main activities: Development of bio-, immuno- and DNA-sensors;Development of bio-analytical methods for the food, environmental analysisand biomedical application; Synthesis and application of conducting polymers.

NanoTechnas,Faculty of Chemistry, Vilnius University,Naugarduko 22, 2006 Vilnius, Lithuania.e-mail: [email protected].

development of biosensors -...

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